Academic literature on the topic 'Germline ETV6'

Abstract Recent studies have identified germline mutations in TP53, PAX5, ETV6, and IKZF1 in kindreds with familial acute lymphoblastic leukemia (ALL), but the genetic basis of ALL in many kindreds is unknown despite mutational analysis of the exome. Here, we report a germline deletion of ETV6 identified by linkage and structural variant analysis of whole-genome sequencing data segregating in a kindred with thrombocytopenia, B-progenitor acute lymphoblastic leukemia, and diffuse large B-cell lymphoma. The 75-nt deletion removed the ETV6 exon 7 splice acceptor, resulting in exon skipping and protein truncation. The ETV6 deletion was also identified by optimal structural variant analysis of exome sequencing data. These findings identify a new mechanism of germline predisposition in ALL and implicate ETV6 germline variation in predisposition to lymphoma. Importantly, these data highlight the importance of germline structural variant analysis in the search for germline variants predisposing to familial leukemia.

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer in children, and the etiology of this aggressive cancer is not fully understood. Common germline polymorphisms in lymphoid development genes and tumor suppressor genes have been associated with ALL susceptibility, although most have modest effects. Only a small fraction of ALL cases are thought to be related to congenital genetic disorders and consequently hereditary predisposition is rarely considered in clinical practice. However, a growing number of rare germline genetic mutations have been discovered in familial ALL (e.g., PAX5, TP53), raising the possibility that the proportion of ALL attributable to inherited predisposition may be higher than currently proposed. In particular, germline ETV6 variations were recently reported in families with hereditary thrombocytopenia and dramatically increased susceptibility to hematologic malignancies (Nat Genet 2015 47: 180 and 535). ETV6 is a transcriptional repressor essential for hematopoiesis and is frequently targeted by somatic genomic aberrations in childhood ALL (e.g., the ETV6-RUNX1 fusion). Therefore, we sought to comprehensively identify ALL predisposition variants in ETV6 and to determine the extent to which these variants contribute to childhood ALL risk in general. We first identified a family with three cases of childhood ALL at St. Jude Children's Research Hospital. Whole exome sequencing of this family (mother and 2 daughters with ALL, the unaffected father and 1 unaffected daughter) identified a single variant in ETV6 (p.R359X) in the 3 cases with ALL and also in the healthy daughter. This nonsense variant is predicted to create a stop codon within the ETS domain of ETV6, resulting in a truncated protein without DNA-binding function. This highly damaging variant is likely to be responsible for the ALL predisposition in this family with a high albeit incomplete penetrance. To comprehensively determine the prevalence of ALL-predisposing alleles in ETV6, we performed targeted sequencing of this gene in 4,405 children with newly-diagnosed ALL enrolled on the Children's Oncology Group (COG) AALL0232, P9904, P9905 and P9906 protocols and St. Jude Total Therapy XIIIA, XIIIB and XV studies. We identified a total of 43 germline variants in the exonic regions of ETV6. Thirty-one of the 43 ETV6 variants were defined as "ALL-related" because they were not found or extremely rare in non-ALL populations (N=60,706). These ALL risk variants included 4 nonsense, 21 missense, 1 splice site, and 5 frameshift variants occurring in 35 children (0.79% of ALL cases studied). Fifteen of the 31 ALL-relatedvariants (48.4%) were clustered in the ETS DNA-binding domain of ETV6. We used the combined annotation dependent depletion algorithm (CADD) to predict deleterious effects of each variant. ALL-related ETV6 variants were significantly more likely to be damaging compared to germline variants observed in the non-ALL population (mean CADD phred-like score of 25.6 vs 15.2, respectively, p<0.0001). Interestingly, of the 18 most deleterious ETV6 variants, 10 (55.6%) resided in the ETS domain although none were located within the helix directly interacting with target DNA. Instead, 7 of the 10 variants in ETS domain were between the first and second helices. We next analyzed the relationship between germline risk variants in ETV6 and clinical features of ALL in a subset of 2,021 cases enrolled on St. Jude and COG frontline ALL trials. These cases were comprehensively evaluted for ALL charateristics and representative of the US childhood ALL population. Children with ALL-related ETV6 variants were significantly older at the time of diagnosis than those without these variants (9.5 years vs 6.4 years; P=0.009). The hyperdiploid leukemia karyotype was strikingly overrepresented in ALL cases harboring germline ETV6 risk variants compared to the wildtype group (64.3% vs 26.8%; P=0.0045). In contrast, the frequency of somatic ETV6 -RUNX1 fusion was much lower in cases with ETV6 germline risk variants, compared to cases with wildtype ETV6 (7.1% vs 22.7%), even though this difference did not reach statistical significance. Of note, there was also a trend towards overrepresentation of females in carriers of ALL-related ETV6 variants (71.4% vs 45.7%; P=0.063). In conclusion, our findings indicate that germline ETV6 variations are important determinants for genetic predisposition to childhood ALL. Disclosures Martin: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gentium SpA/Jazz Pharmaceuticals: Research Funding. Evans:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Hunger:Spectrum Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Merck: Equity Ownership; Sigma Tau: Consultancy.

Key PointsWe report the first known family with a constitutional translocation disrupting ETV6 predisposing to ALL. Germline monoallelic expression of ETV6 contributes to leukemia predisposition without thrombocytopenia.

Abstract There is increasing evidence for inherited susceptibility to acute lymphoblastic leukemia (ALL) in children. Both common and rare germline genetic variants can significantly influence the risk of developing ALL, often in an age- and/or leukemia subtype-dependent manner (e.g., ARID5B, IKZF1, GATA3, PIP4K2A, PAX5, and TP53). Recently, we and others reported a novel genetic syndrome characterized by damaging germline ETV6 mutations that co-segregate with hereditary thrombocytopenia and increased propensity for hematologic malignancies (especially ALL). Sequencing 4,405 childhood ALL cases, we subsequently identified 30 germline coding ETV6 variants that were potentially responsible for disease predisposition in 35 patients (Lancet Oncol 2015). These findings unequivocally point to ETV6 as a critical ALL risk gene; however, the exact functional consequences of the ALL-related ETV6 variants are largely unknown and the mechanism by which they contribute to leukemogenesis is unclear. To this end, we first systematically evaluated these 30 ETV6 risk variants for their transcriptional repressor activity, ability to bind to target DNA sequences, localize to the nucleus, and also to homo-dimerize. Using PF4 and MMP3 promoter-driven luciferase transcription in HEK293T cells as the model system, we showed that 18 of these 30 ETV6 variants had loss of transcriptional repression activity (median of 53.2% of wildtype ETV6 activity [range 7.1% to 83.9%]). These include 4 of the 5 frameshift variants, all 4 nonsense variants, and also 10 of 11 missense variants in the ETS/DNA binding domain. None of the missense variants outside the ETS domain had significant effects on transcriptional repression function, and surprisingly the E44fs variant showed enhanced activity. Co-expression of wildtype and each of the 18 loss-of-function variants consistently impaired the normal repressor activity of wildtype ETV6, indicating dominant-negative effects. In cells expressing both wildtype and variant ETV6, immunoprecipitation of variant protein pulled down wildtype ETV6, in line with the dominant negative mode of action of variant ETV6 via dimerization with wildtype protein. Cellular fractionation showed significantly increased cytoplasmic localization of 18 loss-of-function ETV6 variants when expressed in HEK293T cells with concomitant reduction in nuclear ETV6. Analysis using EMSA revealed that all 14 variants in the ETS domain resulted in impaired binding to the consensus sequence in ETV6 target genes in vitro, as was also true for all 4 loss-of-function frameshift variants. To examine the role of ETV6 variants on hematopoietic stem and progenitor cell function, we lentivirally transduced murine LSK cells with wildtype or variant murine Etv6 (R355X or R395C). These cells were cultured ex vivo and analyzed for myeloid and pre-B cell differentiation, using colony formation assay. We found no change in the formation of myeloid colonies, whereas the number of pre-B colonies increased slightly with mutant Etv6, although we observed no effects on cell cycle or apoptosis. Focusing on the 18 ETV6 variants with experimentally confirmed deleterious effects, we then evaluated the association of germline ETV6 status with ALL clinical features in 3,906 children from frontline ALL protocols at St. Jude Children's Research Hospital and Children's Oncology Group. Patients with these ETV6 variants were significantly older at ALL diagnosis than those without (13.3 [2.6-21.7] vs 6.8 [0.08-30.7] years, P=0.026) but were more likely to have leukocyte count <50x109/L at presentation (100% vs. 68.6%, P=9.5x10-4). 80% of patients with deleterious ETV6 variants had hyperdiploid ALL compared to 22.2% of those with normal ETV6 (P=9.3x10-8), strongly suggesting interactions between germline and somatic variations during leukemogenesis. Finally, whole genome sequencing of matched ALL blast and germline samples from 5 familial ALLs in 2 kindreds with pathologic ETV6 variants identified somatic lesions as plausible second events to promote leukemogenesis in these cases. In conclusion, we comprehensively characterized 30 ALL risk variants in ETV6, the majority of which function by disrupting DNA binding and impairing its transcriptional repressor activity in a dominant negative fashion. ETV6 variants also affect hematopoiesis but most likely require additional somatic lesions for overt leukemogenesis. Disclosures Martin: Novartis: Other: Support of clinical trials; Jazz Pharmaceuticals: Other: One time discussion panel. Evans:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping.. Relling:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Hunger:Patent: Patents & Royalties: Dr. Hunger is a co-inventor of a patent (#8658,964) for the identification of novel subgroups in high risk B-ALL and outcome correlations and diagnostic methods related to the same; Erytech: Honoraria; Sigma Tau Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Honoraria; Merck: Equity Ownership; Pfizer: Equity Ownership; Amgen: Equity Ownership; Spectrum Pharmaceuticals: Honoraria. Mullighan:Loxo Oncology: Research Funding; Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees.

Abstract Recent studies from our group and others have revealed a role for ETV6 germline mutations in the predisposition to ALL. Although ETV6 is among the most commonly mutated genes in ALL, its mechanistic role in leukemogenesis remains unclear. ETV6 is an ETS family transcription factor. ETV6 regulates gene transcription through homo- and hetero- oligomerization with other ETS family members and transcriptional repressors. The germline mutation (P214L amino acid change) identified by our group and others impairs the transcriptional activity and nuclear localization of ETV6 in a dominant negative fashion. The goal of this project is to determine the role of ETV6 in early B cell development and define how germline ETV6 mutations result in predisposition to leukemia. To identify functions of ETV6 in B cell development, we queried the gene expression commons database for evidence of Etv6 expression during B cell development. Etv6 is highly expressed in hematopoietic stem and lymphoid progenitor cells through the pre-pro-B stage (FrA), but its expression is significantly reduced in fraction B and thereafter (P<0.0001). To confirm relative patterns of Etv6 and Pax5 expression in developing B cells, we isolated bone marrow (BM) from wild type (WT) mice and fractionated cells committed to the B cell lineage via B220+ and CD43+ staining by flow cytometry and then separated into the following fractions: Fraction A (CD24low, CD19-), Fraction B (CD19+, CD24+, BP1-) and Fraction C (CD19+ CD24+ BP1+). Etv6 expression decreases as B cells develop and is negatively correlated with Pax5 expression (r2=.9993; P= 0.0167). We next confirmed the expression patterns of ETV6 and PAX5 during B cell development in human samples. We found that ETV6 expression was higher in the early B cell fraction (CD10+, CD34+, CD19-, and CD20-) compared to the preB cell fraction (CD10+, CD34-, CD19+, CD20-). Conversely, we observed that PAX5 expression was higher in the preB cell fraction compared to the early B cell fraction. To determine if a function relationship exists between ETV6 and Pax5 we overexpressed an empty vector (MiG), wild type (WT) ETV6 and ETV6 P214L in a murine lymphoid progenitor line (Ba/F3). ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (P≤0.05). To further interrogate the role of ETV6 in regulating Pax5 transcription we measured the association of ETV6 with putative ETS factor binding sites (GGAA sequence) within the Pax5 transcription start site (TSS) using ChIP-PCR. ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We next determined the consequences of ETV6 mutation on the recruitment of ETV6, SIN3A, and HDAC3 to the Pax5 locus by performing ChIP-PCR in Ba/F3 cells that express a FLAG-tagged WT ETV6 or ETV6 P214L. We detected association of ETV6, SIN3A and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. We conclude that ETV6, SIN3A and HDAC3 are responsible for the repression of Pax5 transcription. Moreover, mutant ETV6 inhibits the ability of normal ETV6 to bind and recruit SIN3A and HDAC3 to the Pax5 locus. Finally, we determined if the recruitment of SIN3A and HDACs to the Pax5 locus was essential to repression of Pax5 by WT ETV6 by knocking out SIN3A and inhibiting HDACs using pan HDAC inhibitor, SAHA and measuring Pax5 expression by RT-PCR. We found that upon SIN3A knockout or HDAC inhibition Pax5 expression was no longer repressed upon WT ETV6 overexpression. To determine the consequences of ETV6 P214L expression on B cell development, we generated a transgenic mouse expressing the P214L mutation in the endogenous ETV6 gene. Preliminary data suggests that these mice have thrombocytopenia, similar to patients with germline ETV6 mutation. In addition, mice with the ETV6 P214L mutation displayed reduced level of cKIT expression on the FrA B cell population. Further studies will be necessary to understand the consequences of reduced cKIT expression to overall B cell development and if this cKIT reduction is linked to aberrant Pax5 expression. In conclusion, ETV6 regulates Pax5 expression through the recruitment of SIN3A and HDAC3 to the Pax5 locus. These findings are significant because Pax5 misregulation results in a B cell development halt, lineage infidelity and leukemogenesis. Disclosures No relevant conflicts of interest to declare.

OBJECTIVES/SPECIFIC AIMS: The goal of this project is to determine the role of ETV6 in early B-cell development and define how germline ETV6 mutations result in predisposition to leukemia. METHODS/STUDY POPULATION: Gene expression commons were queried for expression levels of Etv6 and Pax5 at different stages of hematopoiesis. Mouse bone marrow was isolated and fractioned into cells committed to the B cell lineage via B220+ and CD43+ staining by flow cytometry and then separated into the following fractions: Fraction A (CD24low, CD19−), Fraction B (CD19+, CD24+, BP1−), and Fraction C (CD19+ CD24+ BP1+). Wild-type or germline mutant P214L ETV6 were cloned in an MiG vector and expressed in Ba/F3 cells. ChIP-PCR was performed by cross-linking proteins to DNA with 1% formaldehyde for 10 minute at room temperature, followed by cell lysis with RIPA buffer. Lysates were sonicated to shear DNA to a length of 200–1000 base pairs, then Protein A agarose beads were used to clean and immunoprecipitate chromatin. RESULTS/ANTICIPATED RESULTS: We observed that Etv6 is highly expressed in hematopoietic stem and lymphoid progenitor cells through the pre-pro-B stage (FrA), but its expression is significantly reduced in fraction B and thereafter (p<0.0001). Etv6 expression decreases as B cells develop and is negatively correlated with Pax5 expression (r2=0.9993; p=0.0167). We next confirmed the expression patterns of ETV6 and PAX5 during B cell development in human samples. We found that ETV6 expression was higher in the early B cell fraction (CD10+, CD34+, CD19−, and CD20−) compared to the pre-B cell fraction (CD10+, CD34−, CD19+, CD20−). Conversely, we observed that PAX5 expression was higher in the preB cell fraction compared with the early B cell fraction. In Ba/F3 cells expressing ETV6 constructs, ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (p≤0.05). ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We detected association of ETV6, SIN3A, and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results provide a mechanism of interaction for ETV6 and PAX5, 2 genes often disrupted in B-cell leukemia. These findings are significant because PAX5 misregulation results in a B cell development halt, lineage infidelity, and leukemogenesis. In continuing our studies, we have generated a transgenic mouse endogenously expressing the ETV6 P214L mutation by CRISPR/Cas9 editing, and these mice appear to have a thrombocytopenic phenotype similar to that observed in patients carrying the ETV6 P214L mutation. These animals will be the focus of our continued investigation of the mechanism by which ETV6 germline mutation results in a predisposition to leukemia. Our ultimate goal is a comprehensive understanding of how this process may be targeted more efficiently in patients with both heritable and sporadic forms of leukemia involving ETV6.

Background. The identification of germline mutations in familial leukemia predisposition genes by next generation sequencing is of pivotal importance. Lately, some “blend pedigrees” characterized by both solid and hematologic malignancies have been described. Some genes were recognized as related to this double predisposition, while the involvement of others is still a matter of debate. ETV6 was associated with hematologic malignancies, in particular myeloid malignancies, and recently described as mutated also in oncologic patients. No clear evidences in its involvement in blend pedigrees are known. Case Presentation. We present our recent experience in the identification of an ETV6-mutated “blend pedigree,” suggesting the involvement of ETV6 in the predisposition to both solid and hematologic neoplasia. The pedigree recognition started with a MDS case enrolled in the NEXT-Famly protocol. The patient presented 9 relatives affected by solid tumors and hematological malignancies. Following the clinical trial protocol, the patient underwent NGS analysis, which confirmed the presence of a mutation on the noncoding region of ETV6 both on tumor and on germline DNA. The mutation resulted was shared by the still alive affected relatives. Conclusion. This evidence supports the involvement of ETV6 in the predisposition to both solid and hematologic neoplasia and the importance of the investigation of the noncoding regions of the genes as recently suggested by different expert groups.

Abstract Childhood acute lymphoblastic leukemia (ALL) is the most common cancer in children. The overall survival rate has reached to 90%. However, ALL still presents a significant disease burden and is a major cause for deaths in children. Recently, both inherited germline variants related to ALL susceptibility and somatic genetic variants forming novel subgroups of ALL have been discovered. In this thesis two families with familial ALL were studied. Constitutional heterozygous microdeletion at chromosome 7p12.1p13, including IKZF1, was discovered in the first family with intellectual impairment, overgrowth, and susceptibility to childhood ALL. In the second family, constitutional chromosome translocation was revealed in two individuals with childhood ALL and, subsequently, in seven unaffected family members. The balanced reciprocal translocation t(12;14)(p13.2;q23.1) resulted in breakpoints on two genes; ETV6 on chromosome 12 and RTN1 on chromosome 14. Only a few familial and sporadic ALL cases with germline variants in either IKZF1 or ETV6 have been published, thus supporting the significant role of these constitutional variants in childhood ALL predisposition. Inherited bone marrow failure syndromes (IBMFS) may predispose to childhood leukemia, including ALL. Two unrelated patients were diagnosed with bone marrow failure without the symptoms of classical IBMFS. Neither patient had any signs of developmental delay or congenital anomalies. Exome sequencing revealed identical c.1457del(p.(Ile486fs)) mutation on the ERCC6L2 gene in both patients. A few patients with IBMFS and ERCC6L2 variants have been described in previous studies. Some of them also had congenital craniofacial anomalies and developmental delay that were not detected in the patients in this thesis. The ALL cohort study on genetic variation of mitochondrial DNA (mtDNA) included 36 children. Metabolic change where malignant cells uncouple energy production from oxidative phosphorylation (OXPHOS) is one of the established hallmarks of cancer. In the cohort in this study, 22% of patients harbored nonsynonymous variants on mtDNA in the protein-coding genes of OXPHOS enzyme complexes. The somatic non-neutral variants were found in patients with a poor prognosis cytogenetic marker. The results support the hypothesis that cancer cells harbor mtDNA variants that may affect the cell metabolism
Tiivistelmä Akuutti lymfoblastileukemia (ALL) on lasten yleisin syöpä. Vaikka nykyisin noin 90 prosenttia paranee, ALL aiheuttaa huomattavan paljon sairastavuutta ja on merkittävä lasten kuolinsyy. Vastikään on löydetty perinnöllisiä geneettisiä muutoksia, jotka altistavat lapsuusiän ALL:lle. Tutkimuksen kohteena oli kaksi perhettä, joissa vähintään kaksi lasta on sairastunut ALL:aan. Ensimmäisessä perheessä havaittiin lapsuusiän ALL:aan sairastuneilla kehityshäiriöisillä sisaruksilla äidiltä periytyvä heterotsygoottinen deleetio kromosomissa 7p12.1p13, jossa sijaitsee IKZF1-geeni. Toisessa perheessä perinnöllinen kahden kromosomin translokaatio todettiin kahdella lapsuusiän ALL:aan sairastuneella sekä seitsemällä perheenjäsenellä. Balansoitu translokaatio t(12;14)(p13.2;q23.1) aiheuttaa katkaisukohdan ETV6-geeniin kromosomissa 12 ja RTN1-geeniin kromosomissa 14. Tähän mennessä on julkaistu vain muutamia tutkimuksia potilaista, joilla on ollut perinnöllinen muutos joko IKZF1- tai ETV6-geenissä. Näillä geeneillä oletetaan olevan tärkeä merkitys perinnöllisessä alttiudessa sairastua lapsuusiän ALL:aan. Perinnölliset luuytimen toimintahäiriöt voivat altistaa leukemialle, kuten ALL:lle. Kahdella lapsella todettiin luuytimen toimintahäiriö, mutta ei muita oireita, jotka voisivat liittyä tyypillisiin perinnöllisiin luuytimen toimintahäiriöihin. Eksomisekvensoinnissa todettiin identtinen, homotsygoottinen mutaatio c.1457del(p.(Ile486fs)) ERCC6L2-geenissä. Kirjallisuuslähteiden mukaan vain muutamalla potilaalla on todettu ERCC6L2-geenin muutoksesta johtuva luuytimen toimintahäiriö. Osalla heistä on ollut synnynnäisiä kallon ja kasvojen anomalioita sekä kehityshäiriö, jollaisia tähän tutkimukseen osallistuneilla potilailla ei todettu. Potilaskohorttitutkimuksessa tutkittiin mitokondriaalisen DNA:n (mtDNA) muutoksia ALL:aan sairastuneilla lapsilla. Syöpäsolut eivät hyödynnä mitokondrion elektroninsiirtoketjua energian tuotantoon, ja tämä aineenvaihdunnan muutos on tunnustettu syövän ominaisuus. Tutkimuksessa havaittiin, että 22 prosentilla potilaista ilmeni diagnoosivaiheessa poikkeavia mtDNA:n muutoksia, jotka olivat elektroninsiirtoketjun entsyymien alayksiköitä koodaavissa geeneissä. Muutoksia todettiin useimmiten potilailla, joilla oli leukemiasoluissa huonon ennusteen geneettinen tekijä. Havaitut muutokset voivat mahdollisesti vaikuttaa leukemiasolun energia-aineenvaihduntaan